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This study investigates Kalman smoothing (KS)-based frequency-domain channel estimation for uplink multiple-input multiple-output (MIMO) orthogonal frequency division multiple access (OFDMA) systems with time-varying channels. The proposed KS channel estimation scheme significantly outperforms the recursive least squares (RLS) channel estimation in the high signal-to-noise ratio (SNR) range, because of more effective exploitation of the signal information. In addition, channel interpolation is employed to improve the channel estimation accuracy by exploiting the correlation between adjacent subcarriers. The proposed KS channel estimator can also achieve a bit error rate (BER) performance which is close to the case with perfect channel state information (CSI) with a training overhead of only 5%.